1. Field of the Invention
The present invention relates to a new method for purifying factor VIII from cryoprecipitate of human or animal blood, as well as to the preparations obtained by this method.
2. Description of Related Art
The importance of attempting to obtain, in good yields, the purest possible fractions, in order to avoid the presence of contaminants such as other blood proteins or foreign products such as antigens, viruses and the like, is known.
Lundblad et al. (Thrombosis Research, 1, p. 197-200, 1972) purify bovine factor VIII by ion exchange chromatography on a column of TEAE-cellulose in 50 mM Tris buffer, pH 8.6, and an NaCl gradient. They obtain a yield of 15 to 45% and a specific activity multiplied by 25 to 50. The addition of 50 mM glucose to the buffer increases the yield to 60-70% for a specific activity substantially identical to the above.
Vehar and Davie (Biochemistry, 19 (3), p. 401-410, 1980) purify bovine factor VIII on DEAE-Sephadex in 20 mM imidazole buffer, pH 6.6, also containing: 0.15 M NaCl, 10 mM CaCl.sub.2, 20 mM glycine ethyl ester, 10% glycerol, and dithiothreitol. The yield is 75% and the specific activity is multiplied by 103.
Faure et al. (Journal of Chromatography 257, p. 387-391, 1983) purify human factor VIII on an anion exchanger having hydrophobic properties due to hexyl residues (AH-SEPHAROSE). The solution to be purified is a cryoprecipitate extract treated with alumina gel. Using a 0.1 M/0.1 M acetate/lysine equilibration buffer, pH 5.5, the yield is 34% for a specific activity multiplied by 7.7. The addition of sucrose (10 g/l) and albumin (10 g/l) to this buffer is said to increase the yield.
In European Patent Application EP-A-0,173,242, a method is described for purifying factor VIII, in which a cryoprecipitate extract treated with alumina gel undergoes chromatography on an anion exchanger of the polysaccharide type at an acid pH of between 5 and 6.5 and preferably 5.5. This method is noteworthy in that, prior to the chromatography, the cryoprecipitate extract is pasteurised and in that this chromatography is performed in the presence of sucrose and glycine at high concentrations.
U.S. Pat. No. 4,743,680 recommends, on the one hand the use of sugars and polyhydric alcohols to increase the electrostatic forces at the surface of the factor VIII protein and decrease the hydrophobicity during chromatography on an ion exchange column, and on the other hand the use of a surfactant such as Tween 80 in the chromatographic elution buffer. Suggested supports are QAE-SEPHADEX A-25 and QAE-SEPHAROSE 4B-Fast Flow (Pharmaci, Sweden) .
U.S. Pat. No. 4,758,657 proposes the purificatication of factor VIII on a hydrophobic support which is not an anion exchanger, and the use of an elution buffer comprising surfactants.
From European Patent Application EP-A-0,343,27, a method is also known for purifying factor VIII, in which, principally, a cryoprecipitate extract is treated with alumina gel at a temperature of between 10.degree. and 18.degree. C. and is subjected to chromatography on a hydrophilic anion exchange column ( for example on DEAE-FRACTOGEL). The method comprises a viral inactivation step, using a solvent/detergent system (for example TWEEN/TNBP) which can then be removed before the chromatography step by oil extraction. In addition, this document explains that it is essential to perform the extraction of the cryoprecipitate in a medium containing heparin. A similar treatment is described in Patent Application EP-A-0,367,840.
International Patent Application WO 89/12,065 describes a method which is based substantially on the above ( EP-A-0,343,275). This document lays stress, however, on the choice of the chromatography resin so as to permit separation on a single chromatography column and avoid subsequent treatments, such as ultrafiltration, which, according to this application, increase the complexity of the methods and decrease the activity of the protein. The choice of resin settles on a gel of vinyl polymer containing DEAE groups, with slightly hydrophobic properties and having a special capacity to adsorb the very large factor VIII/yon Willebrand factor complexes. FRACTOGEL TSK-DEAE 650 (M) (Merck) complies with this definition and hence proves markedly superior to other types of gels such as DEAE-SEPHAROSE CL-6B, DEAE-SEPHAROSE CL-6B Fast Flow (Pharmacia), DEAE-SEPHAROSE 4B and DEAE-Trisacryl LS (IBF), which are not recommended.
This document also recommends the use of glycine and lysine in the elution buffer and avoids the use of calcium chloride in this buffer. In addition, inactivation with solvent/detergent is optional and may be carried out at any stage of the method. The chromatographic yield is between 75 and 90% and the specific activity of the factor VIII is greater than 100 IU/mg.